Raf Kinase Inhibitory Protein (RKIP), a member of the phosphatidylethanolamine binding protein (PEBP) family, is representative of a new class of modulators of signaling cascades that function to maintain the yin yang or balance of biological systems. An evolutionarily conserved protein that is expressed from bacteria to man, RKIP inhibits MAP kinase (Raf-MEK-ERK), G protein-coupled receptor (GPCR) kinase and NFkB signaling cascades. Because RKIP targets different kinases dependent upon its state of phosphorylation, RKIP also acts to integrate crosstalk initiated by multiple environmental stimuli. Loss or depletion of RKIP results in disruption of the normal cellular stasis and can lead to chromosomal abnormalities and disease states such as cancer. Recent studies from our laboratory and others have revealed a new mechanism for the regulation of the Raf-1-activated MAP kinase cascade by RKIP. Under non-stimulatory conditions, Raf-1 can form a complex with Raf Kinase Inhibitory Protein (RKIP/PEBP). We demonstrated that RKIP interaction with Raf-1 prevents phosphorylation by Raf-1-activating kinases, inhibiting downstream signaling by MAP kinase. We have shown that growth factors activate members of the protein kinase C (PKC) family that, in turn, cause release of RKIP, enabling activation of Raf-1 and the MAPK signaling cascade. Phospholipid binding to RKIP also prevents it from interacting with and inactivating Raf-1. Inhibition of Raf-1 by RKIP is regulated via a novel mechanism we identified involving the highly conserved RKIP lipid binding pocket. Our recent results have implicated RKIP in maintaining cell cycle integrity. For example, we have shown that RKIP regulates Aurora B kinase and the spindle checkpoint via the MAP kinase cascade. Since RKIP has been implicated as a metastatic tumor suppressor, these results provide one mechanism by which loss of RKIP leads to genomic instability and cancer progression. Finally, recent results suggest that RKIP regulation of the GPCR signaling cascade is dependent upon RKIP phosphorylation. We therefore hypothesize that RKIP is a key integrator of cellular signals and a regulator of genomic stability and cancer progression. The goal of this proposal is to characterize further the physiological functions of RKIP, the mechanisms by which RKIP functions, and the structural interactions that underlie these mechanisms. The studies proposed here will increase our understanding of the regulation of these key events in cell proliferation, and should lead to the development of new reagents that can inhibit cell growth and tumor progression. Specifically, we plan to: 1) Determine the mechanism(s) by which RKIP functions as a checkpoint regulator of MAP kinase signaling; 2) Determine the mechanism by which RKIP mediates crosstalk between MAPK and G protein-coupled receptor (GPCR) signaling; and 3) Characterize the structure/function relationships underlying RKIP regulation of the Raf-1/MAPK and GPCR signaling cascades. PUBLIC HEALTH RELEVANCE: Due to its critical position in the MAP kinase signaling module kinase upstream of MAP kinase kinase (MEK) and MAP kinase (ERK) and because of the complexity of its activation, Raf is a key point of control for regulators of the MAP kinase pathway. The study of proteins such as RKIP that regulate Raf will provide great insight into how cells choose between cell division and differentiation; misexpression or disregulation of proteins such as RKIP could lead to inappropriate signaling and, ultimately, cancer. The studies proposed here will increase our understanding of the regulation of this important pathway, and should lead to the development of new reagents that can inhibit tumor growth and potentiate tumor death.